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WO2015066708A1 - Quantification et suivi spatio-temporel d'une cible à l'aide d'un acide nucléique sphérique - Google Patents

Quantification et suivi spatio-temporel d'une cible à l'aide d'un acide nucléique sphérique Download PDF

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Publication number
WO2015066708A1
WO2015066708A1 PCT/US2014/063921 US2014063921W WO2015066708A1 WO 2015066708 A1 WO2015066708 A1 WO 2015066708A1 US 2014063921 W US2014063921 W US 2014063921W WO 2015066708 A1 WO2015066708 A1 WO 2015066708A1
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Prior art keywords
polynucleotide
nanoparticle
target
rna
oligonucleotides
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Inventor
Chad A. Mirkin
William E. BRILEY
Pratik S. RANDERIA
Nathaniel J. KIM
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Northwestern University
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Northwestern University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag

Definitions

  • the present invention relates to methods of detecting and tracking a target molecule using a nanoparticle wherein the nanoparticle comprises a polynucleotide that can
  • RNA Ribonucleic acid
  • mRNA sequences relies not only on proper quantities of mRNA expression, but also the active transport of transcripts to subcellular compartments where highly localized translation can occur [Jansen, Nat Rev Mol Cell Biol 2: 247 (2001)].
  • Beta-actin localizes at the leading lamellae of growing fibroblasts, driving cell motility [Oleynikov et al., Current Biology 13: 199 (2003)].
  • Unfortunately despite the importance of these two aspects in mRNA function, there is no tool available to both measure intracellular concentration and observe localization of mRNA in live cells.
  • NF NanoFlare
  • SNA Spherical Nucleic Acid
  • FISH Fluorescence In Situ Hybridization
  • fixation and permeabilization of cells prior to analysis.
  • analysis of dynamic RNA distribution is restricted to a single snapshot in time.
  • understanding the translocation of RNA with respect to time, cell cycle, or external stimulus is difficult or impossible.
  • fixed cell analysis is a highly specialized procedure, due to the number steps necessary to prepare a sample. Fixation, permeabilization, blocking, and staining processes each require optimization and vary based on cell type and treatment conditions, rendering FISH prohibitively complicated in many cases.
  • live cell analysis platforms such as molecular beacons require harmful transfection techniques such as microinjection or lipid transfection, and are rapidly sequestered to the nucleus upon cellular entry.
  • a new type of analysis platform is required.
  • compositions and methods for determining the intracellular concentration of a target molecule and/or spatio-temporally tracking the target molecule comprising contacting a target polynucleotide with a composition comprising a nanoparticle under conditions that allow association of the target polynucleotide with the nanoparticle, the nanoparticle comprising a first polynucleotide attached thereto, wherein a portion of the first polynucleotide comprises a sequence that is identical to a portion of the target polynucleotide, the nanoparticle further comprising a second polynucleotide, wherein the second polynucleotide: (i) comprises a marker; and (ii) is hybridized to the first polynucleotide; wherein association of the target polynucleotide and the nanoparticle results in: (i) release of the second polynucleotide from the nanoparticle; and (ii) association of the second polynucleotide
  • the position of the signal is determined.
  • the detectable signal is measured at time X and at time Y, wherein time Y is subsequent to time X.
  • the position of the signal is determined at time X and at time Y.
  • the change in position between time X and time Y is determined.
  • the detectable signal is measured in vitro, while in other embodiments, the detectable signal is measured in vivo. In related embodiments, the detectable signal is measured in a cell and/or a tissue. In further embodiments, the cell and/or tissue is fixed. In still further embodiments, the fixed cell and/or tissue is permeabilized. In yet additional embodiments, the cell and/or tissue is fixed and permeabilized.
  • the first polynucleotide and/or the second polynucleotide is DNA. In some embodiments, the first polynucleotide and/or the second polynucleotide is RNA.
  • the marker in various embodiments, is quenched when the second polynucleotide comprising the marker is hybridized to the first polynucleotide.
  • the second polynucleotide comprises a marker which is a detectable label, wherein the marker is detectable only when the second polynucleotide is associated with the target polynucleotide.
  • the nanoparticle in some embodiments, comprises a multiplicity of first polynucleotides and a multiplicity of second polynucleotides.
  • each polynucleotide in the multiplicity of second polynucleotides associate with the same target polynucleotide.
  • at least one polynucleotide in the multiplicity of second polynucleotides associates with a different target polynucleotide than at least one other polynucleotide in the multiplicity of second polynucleotides.
  • the target polynucleotide is a non-coding RNA
  • the non-coding RNA is a piwi-interacting RNA (piRNA).
  • the composition further comprises a therapeutic agent.
  • the composition further comprises a regulatory polynucleotide.
  • the regulatory polynucleotide in various embodiments, is selected from the group consisting of small interfering RNA (siRNA), piwi- interacting RNA (piRNA), and microRNA (miRNA).
  • the first polynucleotide is between about 5 and about 30 bases in length.
  • the second polynucleotide is between about 10 and about 60 bases in length.
  • the second polynucleotide in various embodiments, hybridizes over the entire length of the first polynucleotide. In some embodiments, the second polynucleotide hybridizes over the entire portion of the first polynucleotide that is the same sequence as at least a portion of the target polynucleotide. In further embodiments, hybridization of the second polynucleotide to the first polynucleotide results in an overhang of the second polynucleotide, wherein the overhang is from about 2 to about 30 nucleotides in length.
  • the nanoparticle in further embodiments, comprises about 10 second
  • the difference in melting temperature (T m ) between the first polynucleotide and the second polynucleotide is about 20-25 °C.
  • Figure 1 is a schematic depicting operational differences between Nanoflare and Stickyflare.
  • Nanoflare top
  • the nanoparticle binds to oligonucleotide target and releases the nanoflare to float freely while the target remains bound to the nanoparticle.
  • flares from the stickyflare bottom
  • Figure 2 depicts a characterization of Stickyflare target recognition
  • FIG. 3 shows RNA localization in Mouse Embryonic Fibroblasts, ⁇ -actin- targeting Stickyflares localize to the growth cone of growing lamellae (arrows in the upper middle and left panels), where ⁇ -actin RNA is found. In contrast, Stickyflares targeting the Ul nuclear RNA localize to the nucleus.
  • Figure 4 depicts dynamic ⁇ -Actin mRNA transport in MEF cells. Endogenously expressed ⁇ -Actin mRNA is transported distally towards the growth cone. Dashed boxes indicate the labeled RNA being tracked. Each panel indicates a 50 second advancement and consists of a bright field and a fluorescent image. Cy5-labeled Stickyflare appear as bright spots in the fluorescent images.
  • Figure 5 shows that ⁇ -Actin mRNA colocalized with mitochondria in HeLa cells.
  • Figure 6 depicts the detection of nucleic acid targets.
  • Figure 7 shows the intracellular localization of KRAS mRNA.
  • the present disclosure is directed to a nanoparticle-polynucleotide conjugate, termed the Stickyflare (SF), which enables facile quantification of RNA expression in live cells, and spatio-temporal analysis of RNA transport and localization.
  • SF nanoparticle-polynucleotide conjugate
  • Such a platform allows for, inter alia, the quantification of transcript expression, and the ability to track RNA in real-time in a single cell, without the need for transfection agents or specialized techniques.
  • the Stickyflare was derived from the successful architecture of the Nanoflare (see U.S. Patent Number 8,507,200, incorporated by reference herein in its entirety), and is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence- specific manner.
  • the Nanoflare comprises a 13 nanometer (nm) gold nanoparticle core functionalized with a densely packed, highly oriented shell of oligonucleotides designed to be antisense to a target RNA transcript.
  • a fluorophore- conjugated reporter strand termed the flare
  • Hybridization of the flare holds the fluorophore in close proximity to the gold core of the SNA, effectively quenching
  • Stickyflare transfers a detectable marker-conjugated reporter to the transcript, resulting in a "turning on” of the detectable marker in a quantifiable manner, and the labeling of targeted transcripts, allowing the RNA to be tracked via microscopy as it is transported throughout the cell.
  • This SNA is used, in various aspects, to analyze the expression level and spatial distribution of mRNA in a cell and to observe the real-time transport of the mRNA. Further, the disclosure also allows for the tracking of transcripts that undergo more extensive compartmentalization.
  • the StickyFlare allows for spatio-temporal tracking of target mRNA in live cells.
  • the nontoxic nature of the SNA construct allows for real-time observation of dynamic RNA movement [Massich et al., Mol Pharm 6: 1934 (2009); Massich et al., ACS Nano 4: 5641 (2010)].
  • the Nanoflare architecture does not lend itself well to the tracking of a target molecule such as RNA.
  • the short flares in the NF technology do not just release and then float freely— they are relatively short polynucleotides and they can therefore bind nonspecifically to many off-target molecules. Thus, the flare would be released and as soon as it came into contact with any off-target molecules it would bind on to them and track the non-target molecule.
  • markers such as fluorophores can be delivered into the cytoplasm of cells in high concentrations without disrupting cellular function. This is a marked improvement on molecular beacon technology, which must be microinjected in order to be present at sufficient concentrations.
  • the SNA architecture is resistant to nucleases, meaning lower background fluorescence from degraded marker-containing nucleotides.
  • the SNA architecture triggers virtually no immune response, meaning RNA localization is determined without interruption to cellular function.
  • Hybridization to a target sequence using antisense DNA is significantly more specific when the DNA is present in the SNA structure, compared to free DNA.
  • Stickyflare and spherical nucleic acid refer to a polynucleotide- functionalized nanoparticle as described in the disclosure.
  • the term “specifically recognizes” or “specifically associates” means that a polynucleotide can identify and/or interact with a target molecule with a higher affinity and/or avidity compared to a non-target molecule.
  • T m Melting temperature
  • the Stickyflare is bound to the particle and, as a result, is not sequestered in the nucleus.
  • the Nanoflare technology has already demonstrated that when released to float freely, single stranded flares do not go into the nucleus.
  • the sequestration of molecular beacons indicates that double- stranded DNA oligonucleotides are recognized and actively transported.
  • the DNA duplex of the Stickyflare would be similarly recognized and transported, were it not for the fact that the nanoparticle that it is attached to is many times larger than anything that is allowed into the nucleus.
  • the high local salt concentration around the nanoparticle likely inhibits the recognition of DNA duplexes by these proteins.
  • the net effect is that the Stickyflares cannot be transported to the nucleus when bound to the SNA. Once the flare is pulled away from the SNA, however, it is attached to the target molecule and goes wherever the target goes, including into the nucleus if nuclear RNA is targeted.
  • compositions and methods provided herein function under the principle that a polynucleotide is directly or indirectly labeled with a marker, and association of the polynucleotide with a target molecule results in the marker becoming detectable, or more detectable. Accordingly, when the polynucleotide is not associated with the target molecule, the marker is relatively undetectable, or quenched. While it is understood in the art that the term “quench” or “quenching” is often associated with fluorescent markers, it is contemplated herein that the signal of any marker is quenched when it is relatively undetectable. Thus, it is to be understood that methods described and/or exemplified throughout this description that employ fluorescent markers are provided only as single embodiments of the methods contemplated, and that any marker that can be quenched may be substituted for the exemplary fluorescent marker.
  • a marker as disclosed herein is a label attached directly to the second polynucleotide, this second polynucleotide having a lower binding affinity or binding avidity for the first polynucleotide that is functionalized to a nanoparticle, such that association of the target molecule with the second polynucleotide causes the second polynucleotide to be displaced from its association with the first polynucleotide.
  • the marker is present on a second polynucleotide which can hybridize to the first polynucleotide that is functionalized to a nanoparticle in a position such that the marker is in sufficient proximity to the nanoparticle that the nanoparticle exerts its quenching effect.
  • the hybridized and labeled second polynucleotide is displaced from the first polynucleotide, and the quenching effect of the nanoparticle is abated.
  • First polynucleotide is the polynucleotide that is functionalized to the nanoparticle. In one embodiment, the first polynucleotide is from about
  • the first polynucleotide is from about 5 to about 10, or from about 5 to about 8, or from about 10 to about 20, or from about
  • the first polynucleotide is at least about 5, at least about 10, or at least about 20 nucleotides in length. In specific embodiments, the first polynucleotide is or is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • the melting temperature (T m ) of the first polynucleotide is, in various embodiments, from about 25° C to about 50° C, or from about 25° C to about 45° C, or from about 25° C to about 30° C, or from about 30° C to about 50° C, or from about 30° C to about 45° C, or from about 30° C to about 40° C, or from about 30° C to about 35° C, or from about 35° C to about 50° C, or from about 35° C to about 45° C, or from about 35° C to about 40° C, or from about 40° C to about 50° C, or from about 40° C to about 45° C.
  • the T m of the first polynucleotide is about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, or about 50° C.
  • One of skill in the art can routinely determine the T m of a given polynucleotide using, for example, computer software such as the
  • Second polynucleotide is the polynucleotide that is hybridized to the first polynucleotide. As used herein, the second polynucleotide is "flare.” In one embodiment, the second polynucleotide is from about 5 to about 60 nucleotides in length.
  • the second polynucleotide is from about 5 to about 50, or from about 5 to about 40, or from about 5 to about 30, or from about 5 to about 20, or from about 5 to about 10, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20, or from about 20 to about 50, or from about 20 to about 40, or from about 20 to about 30, or from about 30 to about 50, or from about 30 to about 40 nucleotides in length.
  • the second polynucleotide is at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 nucleotides in length.
  • the second polynucleotide is or is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more nucleotides in length.
  • the first polynucleotide and/or the second polynucleotide is DNA, RNA, or any oligonucleotide analogue disclosed herein (including, but not limited to, a locked nucleic acid (LNA), 2'O-Me RNA, a peptide nucleic acid (PNA), or PS-DNA).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • PS-DNA PS-DNA
  • the melting temperature (T m ) of the second polynucleotide is, in various embodiments, from about 25° C to about 80° C, or from about 25° C to about 75° C, or from about 25° C to about 70° C, or from about 25° C to about 65° C, or from about 25° C to about 60° C, or from about 25° C to about 55° C, or from about 25° C to about 50° C, or from about 25° C to about 45° C, or from about 25° C to about 40° C, or from about 25° C to about 35° C, or from about 25° C to about 30° C, or from about 30° C to about 80° C, or from about 30° C to about 75° C, or from about 30° C to about 70° C, or from about 30° C to about 65° C, or from about 30° C to about 60° C, or from about 30° C to about 55° C, or from about 30° C to about 50° C, or from about 30° C to about 45°
  • the T m of the second polynucleotide is about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, about 65° C, about 70° C, about 75° C, or about 80° C. In still further embodiments, the T m of the second
  • polynucleotide is at least about 25° C, at least about 30° C, at least about 35° C, at least about 40° C, at least about 45° C, at least about 50° C, at least about 55° C, at least about 60° C, at least about 65° C, at least about 70° C, at least about 75° C, or at least about 80° C.
  • the second polynucleotide is or is at least 1, is or is at least 2, is or is at least 3, is or is at least 4, is or is at least 5, is or is at least 6, is or is at least 7, is or is at least 8, is or is at least 9, is or is at least 10, is or is at least 11, is or is at least 12, is or is at least 13, is or is at least 14, is or is at least 15, is or is at least 16, is or is at least 17, is or is at least 18, is or is at least 19, is or is at least 20, is or is at least 21, is or is at least 22, is or is at least 23, is or is at least 24, is or is at least 25, is or is at least 26, is or is at least 27, is or is at least 28, is or is at least 29, is or is at least 30, is or is at least 31, is or is at least 32, is or is at least 33, is or is at least 34, is or is at least 35, is or is at least 36, is or is at least 37, is or is at least 38, is or is at least 39
  • polynucleotide is from about 1 to about 50, or from about 1 to about 40, or from about 1 to about 30, or from about 1 to about 20, or from about 1 to about 10, or from 1 to about 5, or from about 5 to about 50, or from about 5 to about 40, or from about 5 to about 30, or from about 5 to about 20, or from about 5 to about 10, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20, or from about 15 to about 50, or from about 15 to about 40, or from about 15 to about 30, or from about 15 to about 20, or from about 20 to about 50, or from about 20 to about 40, or from about 20 to about 30, or from about 30 to about 50, or from about 40 to about 50 nucleotides greater in length relative to the first polynucleotide.
  • the sequences of the first polynucleotide and the second polynucleotide are chosen such that the difference in T m between the first polynucleotide and the second polynucleotide is or is about 20° C.
  • the nucleotide sequence of the first polynucleotide yields a T m of 50° C
  • the nucleotide sequence of the second polynucleotide yields a T m of 70° C, thus resulting in a difference in T m of 20° C.
  • sequences of the first polynucleotide and the second polynucleotide are chosen such that the difference in T m between the first polynucleotide and the second polynucleotide is or is about 5° C, is or is about 10° C, is or is about 15° C, is or is about 25° C, or is or is about 30° C.
  • sequences of the first polynucleotide and the second polynucleotide are chosen such that the difference in T m between the first polynucleotide and the second polynucleotide is or is about 5° C, is or is about 10° C, is or is about 15° C, is or is about 25° C, or is or is about 30° C.
  • the sequences of the first polynucleotide and the second polynucleotide are chosen such that the difference in T m between the first polynucleotide and the second polynucleotide is or is about 5° C, is or is
  • polynucleotide and the second polynucleotide are chosen such that the difference in T m between the first polynucleotide and the second polynucleotide is from about 5° C to about 30° C, or from about 5° C to about 25° C, or from about 5° C to about 20° C, or from about 5° C to about 15° C, or from about 5° C to about 10° C, or from about 10° C to about 30° C, or from about 10° C to about 25° C, or from about 10° C to about 20° C, or from about 10° C to about 15° C, from about 15° C to about 30° C, or from about 15° C to about 25° C, or from about 15° C to about 20° C, or from about 20° C to about 30° C, or from about 20° C to about 25° C.
  • hybridization of the second polynucleotide to the first polynucleotide results in an overhang of the second polynucleotide, wherein the overhang is from about 2 to about 30 nucleotides in length.
  • the overhang is from about 2 to about 25, or from about 2 to about 20, or from about 2 to about 15, or from about 2 to about 10, or from about 2 to about 5, or from about 5 to about 30, or from about 5 to about 25, or from about 5 to about 20, or from about 5 to about 15, or from about 5 to about 10, or from about 10 to about 30, or from about 10 to about 25, or from about 10 to about 20, or from about 10 to about 15, or from about 15 to about 30, or from about 15 to about 25 or from about 15 to about 20, or from about 20 to about 30, or from about 20 to about 25, or from about 25 to about 30 nucleotides in length.
  • hybridization of the second polynucleotide to the first polynucleotide results in an overhang of the second polynucleotide, wherein the overhang is or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the degree of hybridization between the first polynucleotide and the second polynucleotide is over the entire length of the first polynucleotide.
  • the second polynucleotide hybridizes over the entire portion of the first polynucleotide that is the same sequence as at least a portion of the target polynucleotide.
  • the second polynucleotide hybridizes over the entire length of the first polynucleotide that is not part of the spacer sequence as defined herein.
  • the second polynucleotide does not hybridize to the full length of the first polynucleotide. In such embodiments, it is contemplated that the second polynucleotide hybridizes to about 70%, about 80%, about 90%, about 95% or more of the length of the first polynucleotide.
  • the degree of complementarity between the first polynucleotide and the second polynucleotide is contemplated, in various embodiments, to be about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more.
  • SF applications in the nucleus the flare portion of the stickyflare enters the nucleus and, once in the nucleus, is used to bind promoter regions, or proteins involved in transcription or chromatin remodeling, in order to silence gene expression.
  • the Stickyflare could affect the available sites for the spliceosome. This would lead to the ability to control cell differentiation or fate.
  • the present disclosure provides compositions and methods for quantifying and spatio-temporally tracking a target molecule.
  • the target molecule in various embodiments, is selected from the group consisting of an RNA molecule, a DNA molecule, a hybrid RNA:DNA molecule, or a polypeptide.
  • the RNA molecule in various embodiments, is messenger RNA (mRNA), pre-mRNA, micro-RNA (miRNA), or pri-miRNA.
  • the DNA or RNA target molecule is single stranded or double stranded.
  • the second polynucleotide is an aptamer.
  • Methods of detecting the SF include microscopy and flow cytometry.
  • Flow cytometry for quantification- cells are treated with Stickyflares and allowed to interact with the cells for a time sufficient for the Stickyflares to be endocytosed, released into the cytoplasm, and interact with a sample population of the target molecule.
  • this length of time changes depending on the target, cell type, and treatment conditions, but is contemplated to be from about 30 minutes to about 48 hours.
  • Microscopy for quantification and tracking Treatment conditions are the same as those outlined above for flow cytometry.
  • fluorescence microscopy may be used to track the reporter fluorophore.
  • the flares are attached to something other than a fluorescent molecule. In such a case other techniques could be used such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and darkfield microscopy.
  • the Stickyflare is used in vitro in cells and/or tissues that are fixed and permeabilized. In such embodiments, it is contemplated that the Stickyflare enters the cell and/or tissue and labels one or more nucleic acid targets. DETECTABLE MARKER/LABEL
  • a "marker” as used herein is interchangeable with “label” and regardless of the type of interacting compound being identified, methods are provided wherein polynucleotide complex formation is detected by an observable change.
  • complex formation gives rise to a change which is observed with a microscope, such as a fluorescent microscope.
  • markers also include, but are not limited to, redox active probes, other
  • nanoparticles, and quantum dots as well as any marker which can be detected using spectroscopic means, i.e., those markers detectable using microscopy and cytometry.
  • Suitable fluorescent molecules are also well known in the art and include without limitation 1,8-ANS (l-Anilinonaphthalene-8-sulfonic acid), l-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 5-(and-6)-Carboxy-2', 7'-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX
  • 6-TET SE pH 9.0, 7-Amino-4-methylcoumarin pH 7.0, 7-Hydroxy-4-methylcoumarin, 7-
  • Alexa 546 Alexa 555
  • Alexa 568 Alexa 594
  • Alexa 647 Alexa 660
  • Alexa 680 Alexa
  • Alexa Fluor 430 antibody conjugate pH 7.2 Alexa Fluor 488 antibody conjugate pH 8.0
  • TMR-X antibody conjugate pH 7.2 Bodipy TMR-X conjugate, BODIPY TMR-X, MeOH, BODIPY TMR-X, SE, BODIPY TR-X phallacidin pH 7.0, BODIPY TR-X, MeOH,
  • DAPI DAPI-DNA
  • Dapoxyl (2-aminoethyl) sulfonamide DDAO pH 9.0
  • Di-8 ANEPPS Di-
  • 8-ANEPPS-lipid Dil, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed, DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (Enhanced Green Fluorescent Protein),
  • Eosin Eosin antibody conjugate pH 8.0, Erythrosin-5-isothiocyanate pH 9.0, Ethidium
  • Fluorescent Protein FDA, FITC, FITC antibody conjugate pH 8.0, FlAsH, Fluo-3, Fluo-3
  • FM 1-43 lipid FM 4-64, FM 4-64, 2% CHAPS, Fura Red Ca2+, Fura Red, high Ca, Fura
  • LysoSensor Blue LysoSensor Blue pH 5.0
  • LysoSensor Green LysoSensor Green pH 5.0
  • NeuroTrace 500/525 green fluorescent Nissl stain-RNA, Nile Blue, EtOH, Nile Red, Nile
  • Rhod-2 Rhod-2 Ca2+, Rhodamine, Rhodamine 110, Rhodamine 110 pH 7.0, Rhodamine
  • Suitable particles include polymeric particles (such as, without limitation, polystyrene particles, polyvinyl particles, acrylate and methacrylate particles), liposomal particles, glass particles, latex particles, Sepharose beads and others like particles well known in the art. Methods of attaching oligonucleotides to such particles are well known and routinely practiced in the art.
  • chemiluminescent molecules which will give a detectable signal or a change in detectable signal upon hybridization.
  • polynucleotide either functionalized on a SNA or as a target molecule, is used interchangeably with the term oligonucleotide.
  • nucleotide or its plural as used herein is interchangeable with modified forms as discussed herein and otherwise known in the art.
  • base which embraces naturally- occurring nucleotides as well as
  • methods provided include use of polynucleotides which are DNA oligonucleotides, RNA oligonucleotides, or combinations of the two types. Modified forms of oligonucleotides are also contemplated which include those having at least one modified intemucleotide linkage. Modified polynucleotides or oligonucleotides are described in detail herein below.
  • compositions are contemplated which include those wherein a nanoparticle comprises a polynucleotide which further comprises a spacer.
  • the first polynucleotide comprises a spacer.
  • Spacer as used herein means a moiety that serves to increase distance between the nanoparticle and the polynucleotide, or to increase distance between individual
  • polynucleotides when attached to the nanoparticle in multiple copies.
  • the spacer does not directly participate in the activity of the polynucleotide to which it is attached.
  • the spacer is contemplated herein as being located between individual polynucleotides in tandem, whether the polynucleotides have the same sequence or have different sequences.
  • the spacer when present is an organic moiety.
  • the spacer is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, or a combination thereof.
  • the length of the spacer in various embodiments at least about 5 nucleotides, at least about 10 nucleotides, 10-30 nucleotides, or even greater than 30 nucleotides.
  • the spacer may have any sequence which does not interfere with the ability of the polynucleotides to become bound to the nanoparticles or to the second polynucleotide.
  • the bases of the polynucleotide spacer are all adenines, all thymines, all cytidines, all guanines, all uracils, or all some other modified base. MODIFIED OLIGONUCLEOTIDES
  • oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of
  • oligonucleotide oligonucleotide
  • Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates,
  • phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,
  • selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
  • oligonucleotides having inverted polarity comprising a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e. a single inverted nucleoside residue which may be abasic (the nucleotide is missing or has a hydroxyl group in place thereof). Salts, mixed salts and free acid forms are also contemplated.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones;
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones;
  • oligonucleotide mimetics wherein both one or more sugar and/or one or more internucleotide linkage of the nucleotide units are replaced with "non-naturally occurring" groups.
  • this embodiment contemplates a peptide nucleic acid (PNA).
  • PNA compounds the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone. See, for example US Patent Nos. 5,539,082; 5,714,331 ; and 5,719,262, and Nielsen et al , 1991, Science, , 254: 1497-1500, the disclosures of which are herein incorporated by reference.
  • oligonucleotides are provided with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and including— CH 2 — NH— O— CH 2 — ,— CH 2 — N(CH 3 )— O— CH 2 — remind— CH 2 — O— N(CH 3 )— CH 2 — ,— CH 2 —
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Q to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Other embodiments include 0[(CH 2 ) n O] m CH 3 , 0(CH 2 ) n OCH 3 ,
  • n and m are from 1 to about 10.
  • oligonucleotides comprise one of the following at the 2' position: Q to C 10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a modification includes 2'-methoxyethoxy (2'-0-CH 2 CH 2 0CH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, 1995, Helv. Chim. Acta, 78: 486-504) i.e., an alkoxyalkoxy group.
  • modifications include 2'- dimethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 0N(CH3) 2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-0— CH 2 — O— CH 2 — N(CH 3 ) 2 , also described in examples herein below.
  • the 2'-modification may be in the arabino (up) position or ribo (down) position.
  • a 2'-arabino modification is 2'-F.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Pat. Nos.
  • a modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.
  • the linkage is in certain aspects is a methylene (— CH 2 — ) n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
  • Oligonucleotides may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluor
  • Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5 ,4- b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5 ,4- b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases include those disclosed in U.S. Pat. No.
  • bases are useful for increasing the binding affinity and include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. and are, in certain aspects combined with 2'-0-methoxyethyl sugar modifications. See, U.S. Pat. Nos. 3,687,808, U.S. Pat. Nos.
  • a "modified base” or other similar term refers to a composition which can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring base.
  • the modified base provides a T m differential of 15, 12, 10, 8, 6, 4, or 2°C. or less.
  • Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
  • nucleobase is meant the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C 3 — C 6 )- alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-tr- iazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described
  • nucleobase thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by
  • nucleosidic base or “base unit” is further intended to include compounds such as heterocyclic compounds that can serve like nucleobases including certain "universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases.
  • universal bases are 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole), and optionally substituted hypoxanthine.
  • Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.
  • nanoparticle refers to small structures that are less than 10 ⁇ , and preferably less than 5 ⁇ , in any one dimension.
  • nanoparticles contemplated include any compound or substance with a high loading capacity for an oligonucleotide as described herein.
  • a nanoparticle that is functionalized with one or more agents, such as a polynucleotide, is referred to herein as a Spherical Nucleic Acid (SNA).
  • SNA Spherical Nucleic Acid
  • Nanoparticles useful in the practice of the invention include metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials, as long as the nanoparticle has the ability to quench the otherwise detectable marker.
  • metal e.g., gold, silver, copper and platinum
  • semiconductor e.g., CdSe, CdS, and CdS or CdSe coated with ZnS
  • magnetic e.g., ferromagnetite
  • nanoparticles useful in the practice of the invention include ZnS, ZnO, Ti0 2 , Agl, AgBr, Hgl 2 , PbS, PbSe, ZnTe, CdTe, In 2 S 3 , In 2 Se 3 , Cd 3 P 2 , Cd 3 As 2 , InAs, and GaAs.
  • the size of the nanoparticles is preferably from about 5 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm.
  • the size of the nanoparticle is contemplated to be from about 5 to about 10 nm, or about 5 to about 20 nm, or about 5 to about 30 nm, or about 5 to about 40 nm, or about 5 to about 60 nm, or about 5 to about 70 nm, or about 5 to about 80 nm, or about 5 to about 90 nm, or about 5 to about 100 nm, or about 5 to about 110 nm, or about 5 to about 120 nm, or about 5 to about 130 nm, or about 5 to about 140 nm, or about 10 to about 20 nm, or about 10 to about 40 nm, or about 10 to about 50 nm, or about 10 to about 60 nm, or about 10 to about 70 nm, or about 10 to about 80 nm, or about 10 to about 90 nm, or about 10 to about 100 nm, or about 10 to about 110 nm, or about 10 to about 120 nm, or about 10 to about 130 nm, or
  • nanoparticles are contemplated for use in the methods which comprise a variety of inorganic materials including, but not limited to, metals, semi-conductor materials or ceramics as described in US Patent Application No. 20030147966.
  • metal- based nanoparticles include those described herein.
  • Ceramic nanoparticle materials include, but are not limited to, brushite, tricalcium phosphate, alumina, silica, and zirconia.
  • Organic materials from which nanoparticles are produced include carbon.
  • Nanoparticle polymers include polystyrene, silicone rubber, polycarbonate, polyurethanes, polypropylenes, polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, and polyethylene.
  • Biodegradable, biopolymer e.g. polypeptides such as BSA, polysaccharides, etc.
  • other biological materials e.g. carbohydrates
  • polymeric compounds are also contemplated for use in producing nanoparticles.
  • any suitable nanoparticle having molecules attached thereto that are in general suitable for use in detection assays known in the art to the extent and do not interfere with polynucleotide complex formation, i.e., hybridization to form a double-strand or triple-strand complex.
  • the size, shape and chemical composition of the particles contribute to the properties of the resulting oligonucleotide-functionalized nanoparticle. These properties include for example, optical properties, optoelectronic properties, electrochemical properties, electronic properties, stability in various solutions, magnetic properties, and pore and channel size variation.
  • nanoparticles having uniform sizes, shapes and chemical composition is contemplated.
  • suitable particles include, without limitation, nanoparticles, aggregate particles, isotropic (such as spherical particles) and anisotropic particles (such as non-spherical rods, tetrahedral, prisms) and core-shell particles such as the ones described in U.S. Patent
  • Suitable nanoparticles are also commercially available from, for example, Ted Pella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc. (gold).
  • nanoparticles comprising materials described herein are available commercially or they can be produced from progressive nucleation in solution (e.g., by colloid reaction), or by various physical and chemical vapor deposition processes, such as sputter deposition. See, e.g., HaVashi, (1987) Vac. Sci. Technol. July/August 1987, A5(4): 1375-84; Hayashi, (1987) Physics Today, December 1987, pp. 44-60; MRS Bulletin, January 1990, pgs. 16-47.
  • nanoparticles contemplated are produced using HAuCl 4 and a citrate-reducing agent, using methods known in the art. See, e.g., Marinakos et al., (1999) Adv. Mater. 11: 34-37; Marinakos et al., (1998) Chem. Mater. 10: 1214-19; Enustun & Turkevich, (1963) J. Am. Chem. Soc. 85: 3317.
  • Tin oxide nanoparticles having a dispersed aggregate particle size of about 140 nm are available commercially from Vacuum Metallurgical Co., Ltd. of Chiba, Japan.
  • Other commercially available nanoparticles of various compositions and size ranges are available, for example, from Vector Laboratories, Inc. of Burlingame, Calif.
  • a Stickyflare comprises a first polynucleotide and a second polynucleotide, each as described herein.
  • the Stickyflare in various aspects, further comprises a regulatory polynucleotide.
  • the regulatory polynucleotide in various aspects, in various
  • the regulatory polynucleotide is selected from the group consisting of an antisense polynucleotide, short interfering RNA (siRNA), piRNA, or microRNA (miRNA).
  • siRNA short interfering RNA
  • piRNA piRNA
  • miRNA microRNA
  • compositions and methods are therefore contemplated wherein the regulatory polynucleotide is about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, and all polynucleotides intermediate in length of the sizes specifically disclosed to the extent that the regulatory polynucleotide is able to achieve the desired result.
  • polynucleotides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 nucleotides in length are contemplated for the regulatory polynucleotide.
  • Methods for inhibiting gene product expression include those wherein expression of the target gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about
  • the degree of inhibition is determined in vivo from a body fluid sample or from a biopsy sample or by imaging techniques well known in the art. Alternatively, the degree of inhibition is determined in a cell culture assay, generally as a predictable measure of a degree of inhibition that can be expected in vivo resulting from use of a specific type of nanoparticle and a specific polynucleotide.
  • the nanoparticles, the polynucleotides or both are functionalized in order to attach the oligonucleotides to the nanoparticles.
  • Such methods are known in the art.
  • oligonucleotides functionalized with alkanethiols at their 3'-termini or 5'-termini readily attach to gold nanoparticles. See Whitesides, 1995, Proceedings of the Robert A. Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, Tex., pages 109-121.
  • Mucic et al., 1996, Chem. Commun. 555-557 (describes a method of attaching 3' thiol DNA to flat gold surfaces; this method can be used to attach
  • oligonucleotides to nanoparticles can also be used to attach oligonucleotides to other metal, semiconductor and magnetic colloids and to the other nanoparticles listed above.
  • Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for the binding of oligonucleotide -phosphorothioates to gold surfaces), substituted alkylsiloxanes (see, e.g. Burwell, 1974, Chemical Technology, 4: 370-377 and Matteucci and Caruthers, 1981, J. Am. Chem.
  • any suitable method for attaching oligonucleotides onto the nanoparticle surface may be used.
  • a particularly preferred method for attaching oligonucleotides onto a surface is based on an aging process described in U.S. Patent Application No. 09/344,667, filed Jun. 25, 1999;
  • the aging process provides nanoparticle-oligonucleotide conjugates with unexpected enhanced stability and selectivity.
  • the method comprises providing
  • oligonucleotides preferably having covalently bound thereto a moiety comprising a functional group which can bind to the nanoparticles.
  • the moieties and functional groups are those that allow for binding (i.e., by chemisorption or covalent bonding) of the oligonucleotides to nanoparticles.
  • oligonucleotides having an alkanethiol, an alkanedisulfide or a cyclic disulfide covalently bound to their 5' or 3' ends can be used to bind the
  • oligonucleotides to a variety of nanoparticles, including gold nanoparticles.
  • the oligonucleotides are contacted with the nanoparticles in water for a time sufficient to allow at least some of the oligonucleotides to bind to the nanoparticles by means of the functional groups.
  • a time can be determined empirically. For instance, it has been found that a time of about 12-24 hours gives good results.
  • Other suitable conditions for binding of the oligonucleotides can also be determined empirically. For instance, a concentration of about 10-20 nM nanoparticles and incubation at room temperature gives good results.
  • the salt can be any suitable water-soluble salt.
  • the salt may be sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, a combination of two or more of these salts, or one of these salts in phosphate buffer.
  • the salt is added as a concentrated solution, but it could be added as a solid.
  • the salt can be added to the water all at one time or the salt is added gradually over time.
  • gradient over time is meant that the salt is added in at least two portions at intervals spaced apart by a period of time. Suitable time intervals can be determined empirically.
  • the ionic strength of the salt solution must be sufficient to overcome at least partially the electrostatic repulsion of the oligonucleotides from each other and, either the electrostatic attraction of the negatively-charged oligonucleotides for positively-charged nanoparticles, or the electrostatic repulsion of the negatively-charged oligonucleotides from negatively-charged nanoparticles. Gradually reducing the electrostatic attraction and repulsion by adding the salt gradually over time has been found to give the highest surface density of oligonucleotides on the nanoparticles. Suitable ionic strengths can be determined empirically for each salt or combination of salts. A final concentration of sodium chloride of from about 0.1 M to about 1.0 M in phosphate buffer, preferably with the concentration of sodium chloride being increased gradually over time, has been found to give good results.
  • the oligonucleotides and nanoparticles are incubated in the salt solution for an additional period of time sufficient to allow sufficient additional oligonucleotides to bind to the nanoparticles to produce the stable nanoparticle- oligonucleotide conjugates.
  • an increased surface density of the oligonucleotides on the nanoparticles has been found to stabilize the conjugates.
  • the time of this incubation can be determined empirically. A total incubation time of about 24-48, preferably 40 hours, has been found to give good results (this is the total time of incubation; as noted above, the salt concentration can be increased gradually over this total time).
  • This second period of incubation in the salt solution is referred to herein as the "aging" step.
  • Other suitable conditions for this "aging” step can also be determined empirically. For instance, incubation at room temperature and pH 7.0 gives good results.
  • the conjugates produced by use of the "aging” step have been found to be considerably more stable than those produced without the “aging” step. As noted above, this increased stability is due to the increased density of the oligonucleotides on the surfaces of the nanoparticles which is achieved by the "aging” step.
  • An alternative “fast salt aging” process produced particles with comparable DNA densities and stability. By performing the salt additions in the presence of a surfactant, for example approximately 0.01% sodium dodecylsulfate (SDS), Tween, or polyethylene glycol (PEG), the salt aging process can be performed in about an hour.
  • a surfactant for example approximately 0.01% sodium dodecylsulfate (SDS), Tween, or polyethylene glycol (PEG)
  • the surface density achieved by the "aging" step will depend on the size and type of nanoparticles and on the length, sequence and concentration of the oligonucleotides.
  • a surface density adequate to make the nanoparticles stable and the conditions necessary to obtain it for a desired combination of nanoparticles and oligonucleotides can be determined empirically.
  • a surface density of at least 10 picomoles/cm will be adequate to provide stable nanoparticle-oligonucleotide conjugates.
  • the surface density is at least 15 picomoles/cm . Since the ability of the oligonucleotides of the conjugates to hybridize with nucleic acid and oligonucleotide targets can be diminished if the surface density is too great, the surface density is preferably no greater than about 35-40
  • compositions and methods are also provided wherein the oligonucleotide is bound to the nanoparticle at a surface density of at least 2 pmol/cm , at least 5 pmol/cm , at least 10 pmol/cm , at least 15 pmol/cm , at least 20 pmol/cm , at least 25 pmol/cm , at least
  • Hybridization which is used interchangeably with the term “complex formation” herein, means an interaction between two or three strands of nucleic acids by hydrogen bonds in accordance with the rules of Watson-Crick DNA complementarity, Hoogstein binding, or other sequence- specific binding known in the art. Alternatively it can mean an interaction between polypeptides as defined herein in accordance with sequence- specific binding properties known in the art. Hybridization can be performed under different stringency conditions known in the art.
  • hybridization between the two complementary strands or two polypeptides could reach about 60% or above, about 70% or above, about 80% or above, about 90% or above, about 95% or above, about 96% or above, about 97% or above, about 98% or above, or about 99% or above in the reactions.
  • the methods include use of two or three oligonucleotides which are 100% complementary to each other, i.e., a perfect match, while in other aspects, the individual oligonucleotides are at least (meaning greater than or equal to) about 95% complementary to each over the all or part of length of each oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to each other.
  • oligonucleotide used in the methods need not be 100% complementary to each other to be specifically hybridizable. Moreover, oligonucleotide may hybridize to each other over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). Percent complementarity between any given oligonucleotide can be determined routinely using BLAST programs (Basic Local Alignment Search Tools) and PowerBLAST programs known in the art (Altschul et al, 1990, J. Mol. Biol, 215: 403- 410; Zhang and Madden, 1997, Genome Res., 7: 649-656).
  • BLAST programs Basic Local Alignment Search Tools
  • PowerBLAST programs known in the art
  • oligonucleotides on the surface of the nanoparticle is sufficient to result in cooperative behavior between nanoparticles and between polynucleotide strands on a single nanoparticle.
  • the cooperative behavior between the nanoparticles increases the resistance of the oligonucleotide to degradation.
  • stable means that, for a period of at least six months after the conjugates are made, a majority of the oligonucleotides remain attached to the nanoparticles and the oligonucleotides are able to hybridize with nucleic acid and oligonucleotide targets under standard conditions encountered in methods of detecting nucleic acid and methods of nanofabrication.
  • each nanoparticle is functionalized with identical oligonucleotides, i.e., each oligonucleotide attached to the nanoparticle has the same length and the same sequence.
  • each nanoparticle is functionalized with two or more oligonucleotides which are not identical, i.e., at least one of the attached oligonucleotides differ from at least one other attached oligonucleotide in that it has a different length and/or a different sequence.
  • oligonucleotide or “polynucleotide” includes those wherein a single sequence is attached to a nanoparticle, or multiple copies of the single sequence are attached.
  • an oligonucleotide is present in multiple copies in tandem, for example, two, three, four, five, six, seven eight, nine, ten or more tandem repeats.
  • Therapeutic agent as used herein means any compound useful for therapeutic or diagnostic purposes.
  • the terms as used herein are understood to mean any compound that is administered to a patient for the treatment of a condition that can traverse a cell membrane more efficiently when attached to a nanoparticle of the disclosure than when administered in the absence of a nanoparticle of the disclosure.
  • Therapeutic agents useful in the methods of the disclosure include those described in U.S. Patent Application Publication 2012/0282186, which is incorporated by reference herein in its entirety.
  • the present disclosure is applicable to any therapeutic agent for which delivery is desired.
  • active agents as well as hydrophobic drugs are found in U.S. Patent 7,611,728, which is incorporated by reference herein in its entirety.
  • compositions and methods disclosed herein are provided wherein the nanoparticle comprises a multiplicity of therapeutic agents.
  • the multiplicity of therapeutic agents are specifically attached to one nanoparticle.
  • the multiplicity of therapeutic agents is specifically attached to more than one nanoparticle.
  • Therapeutic agents useful in the materials and methods of the present disclosure can be determined by one of ordinary skill in the art.
  • Therapeutic agents include but are not limited to hydrophilic and hydrophobic compounds.
  • Protein therapeutic agents include, without limitation peptides, enzymes, structural proteins, receptors and other cellular or circulating proteins as well as fragments and derivatives thereof, the aberrant expression of which gives rise to one or more disorders. Therapeutic agents also include, as one specific embodiment, chemo therapeutic agents.
  • Therapeutic agents also include, in various embodiments, a radioactive material.
  • Nanoflare necessarily does not allow for investigation of the spatial distribution of targeted RNA. Release of the flare through Nanoflare-transcript binding results in a displacement of the fluorophore from the nanoparticle construct, and thus the transcript, as the RNA remains bound to the antisense capture sequences of the Nanoflare. However, were the complementarity of the Nanoflare oligonucleotides reversed, the result would be a Nanoflare-like construct with the important difference that the flare strands themselves are complementary to, and potentially capable of binding RNA targets.
  • Such a construct may be engineered to perform similarly to the Nanoflare, using base-pair recognition of a target to displace fluorescent flare strands quantifiably, with the additional benefit that the complementary flare remains bound to the RNA ( Figure 1).
  • the Stickyflare we report the development of such a construct, termed the Stickyflare, and investigate its use as a platform for RNA quantification and real-time tracking of transcripts as they are transported within live cells.
  • Oligonucleotides were synthesized using standard solid-phase phosphoramidite chemistry (Expedite 8909 Nucleotide Synthesis System (ABI)). All reagents were purchased from Glen Research. Oligonucleotides were purified by reverse-phase high performance liquid chromatography (HPLC). The oligonucleotide sequences used in this study are shown below. Description Sequence (5' ⁇ 3') SEQ ID NO:
  • alkylthiol-terminated actin and survivin oligonucleotides (3 ⁇ each) were combined with citrate-capped 13 nm gold particles (13 nM) and incubated for 1 hour at room temperature.
  • sodium chloride (NaCl) was added in 0.05M increments over three hours to achieve a final NaCl concentration of 300 mM, and the particles were stored at room temperature for four hours.
  • PBS Phosphate Buffered Saline
  • Flares were hybridized on the purified DNA-gold nanoparticles (DNA-Au NPs) by adding a stoichiometric equivalent of 10 flares/nanoparticle. The solution was then heated to 65 °C and slowly cooled to room temperature overnight to facilitate hybridization. The resulting Stickyflares were then sterilized using a 0.2 ⁇ acetate syringe filter (GE).
  • HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (FBS) (Gibco) supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin. Gene knockdown was performed by treating with 50nM anti-actin siRNA (Santa Cruz
  • RNAiMAX RNAiMAX according to recommended protocol. Cells were then washed once with PBS and further cultured in supplemented OptiMEM.
  • Stickyflare treatment was then performed at 400pM Stickyflares for an additional 24 hours. Fluorescence of trypsinized cells was quantified by using a Guava Easycyte HT flow cytometer (Millipore). Confocal microscopy was performed with Zeiss 510 (Zeiss) and SP5 (Leica) confocal microscopes. Mitochondria were stained using CellLight® Mitochondria- GFP (Life technologies). Evaluation of target recognition by Stickyflares.
  • Stickyflares were first evaluated in vitro for their ability to detect complementary nucleic acid targets.
  • One nanomolar (nM) solutions of ⁇ -actin targeting Stickyflares were evaluated before and after the addition of fully-complementary targets in PBS.
  • nM nanomolar
  • a complementary target Upon addition of a complementary target, a significant increase in fluorescence was observed, indicating displacement of the fluorophore from the nanoparticle surface, while a non- complementary target had no measurable effect (Figure 2a).
  • ⁇ -actin Stickyflares were evaluated in a cell culture model by flow cytometry. HeLa human cervical cancer cells were treated with 50nM of either control or ⁇ -actin siRNA for 24 hours, after which the media was replaced with Stickyflare-containing media.
  • RNA was evaluated using confocal microscopy. Two genes with disparate intracellular function and localization patterns were chosen to analyze spatial distribution within cells: ⁇ -actin mRNA and Ul small nuclear RNA (snRNA). In previous reports, B-actin mRNA has been found to localize at the growth cones of lamellae in embryonic fibroblasts. In contrast, Ul snRNA is imported into the nucleus, where it acts as a key component of the spliceosome.
  • MEFs were cultured in glass-bottomed cell culture chambers with Stickyflares for
  • Stickyflares are not limited to use in live cells, and verifying RNA localization in fixed cells is a convenient control. Therefore, fixed and permeabilized MEFs were treated with
  • the Stickyflare utilizes a targeting strategy that allows for targeting and quantifying RNA targets in live cells, and additionally exploits that recognition event to label target polynucleotides, enabling further analysis of, e.g., RNA transport and
  • this SNA enables a complete analysis of target polynucleotide function in live cells from a single platform, and overcomes many limitations of previous analytical techniques. It is contemplated that the Stickyflare is a valuable tool for investigating, for example and without limitation, proper RNA function and its misregulation in disease, and make such studies accessible to a broader community given the ease of its application in cell culture. Further, the Stickyflare improves analyses in other model systems where asymmetric RNA expression is an essential component, such as, e.g., embryonic development, tissue and organ regeneration, and neurobiology.
  • alkylthiol-terminated actin and survivin oligonucleotides (3 ⁇ each) were combined with citrate-capped 13 nm gold particles (13 nM) and incubated for 1 hour at room temperature.
  • sodium chloride was added to achieve a final concentration of 300 mM, and the particles were stored for four hours.
  • PBS Phosphate Buffer Solution
  • Flares were hybridized on the purified DNA-Au NPs by adding 100 nM (10 flares/NP). The solution was then heated to 65 °C and slowly cooled to room temperature over four hours to allow hybridization. The resulting nanoflares were then sterilized using a 0.2 ⁇ acetate syringe filter (GE Healthcare) to prevent cell contamination and stored at 4°C.
  • GE Healthcare 0.2 ⁇ acetate syringe filter
  • HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (FBS) (Gibco) supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin. Gene knockdown was performed by treating with 50nM anti-survivin siRNA (Santa Cruz
  • NanoFlares A significant increase in fluorescence was observed upon the addition of the complementary target for both constructs (Figure 6, upper left panel), but not observed with the addition of a scrambled target ( Figure 6, upper right panel). This indicated that the SF, like the NF is capable of detecting the presence of nucleic acid targets with sequence- specific discrimination.
  • HeLa cells were subjected to varying amounts of siRNA to knock down the oncogene survivin, then treated with SFs or NFs targeted to that gene. The resultant fluorescence in each cell was evaluated by flow cytometry (Figure 6, lower panel).
  • the StickyFlare is capable of quantifying relative mRNA expression in live cells, and can track changes in the spatial distribution of transcripts over time to gain a more complete understanding of the dynamics of gene expression.

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Abstract

La présente invention concerne des procédés de détection et de suivi d'une molécule cible au moyen d'une nanoparticule, ladite nanoparticule comprenant un polynucléotide pouvant s'associer de façon spécifique avec la molécule cible, et ladite association entraînant une modification touchant un marqueur détectable, modification qui peut être mesurée après l'association avec la molécule cible.
PCT/US2014/063921 2013-11-04 2014-11-04 Quantification et suivi spatio-temporel d'une cible à l'aide d'un acide nucléique sphérique Ceased WO2015066708A1 (fr)

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US10973927B2 (en) 2017-08-28 2021-04-13 The Chinese University Of Hong Kong Materials and methods for effective in vivo delivery of DNA nanostructures to atherosclerotic plaques
US12319711B2 (en) 2019-09-20 2025-06-03 Northwestern University Spherical nucleic acids with tailored and active protein coronae
US11287422B2 (en) 2019-09-23 2022-03-29 Element Biosciences, Inc. Multivalent binding composition for nucleic acid analysis
US12378560B2 (en) 2019-10-29 2025-08-05 Northwestern University Sequence multiplicity within spherical nucleic acids
CN115896261B (zh) * 2022-11-18 2025-08-19 中国地质大学（武汉） 一种基于双嵌段dna探针的球形核酸及其制备方法与应用

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